Liquid for ejection and method for ejecting bio-specimen

ABSTRACT

A liquid for ejection includes a bio-specimen, and at least one kind of first compounds expressed by Formula (1), 
                         
wherein, in Formula (1), m≧8, and 8≦n≦18.

The entire disclosure of Japanese Patent Application No. 2009-153504,filed Jun. 29, 2009 is expressly incorporated by reference herein.

BACKGROUND

1. Technical Field

The present invention relates to liquid for ejection containingbio-specimen and methods for ejecting bio-specimen.

2. Related Art

For examining several ten types of biomolecules contained in blood,several ten cc of blood is necessary at present. In this connection, adetection technology that can substantially reduce the amount of bloodrequired for examination is necessary.

As a method for accurately and efficiently dispensing a very smallamount of liquid, it is possible to use the ink jet technology. Forexample, Japanese Laid-open Patent Application 2008-137987 (PatentDocument 1) describes an example in which a solution containing at leastone kind of protein and peptide is ejected by an inkjet method utilizingthermal energy.

Bio-specimens, such as, blood, contain many molecules, such as, proteinmolecules that would likely adhere nonspecifically to surfaces nearejection orifices and of flow paths of the ink jet head. Therefore,there are cases where those molecules, when adhered, clog the ejectionorifices and the flow paths, incapacitating stable ejection. Also, asthe ejected bio-specimen is subject to biochemical examination, thebioactivity of the contained biomolecules needs to be maintained.However, Patent Document 1 does not describe any concrete methods toaddress such an issue.

SUMMARY

In accordance with some aspects of the invention, there is provided aliquid for ejection containing bio-specimen, which does not lower thebioactivity of biomolecules contained, and is capable of stable ejectionthrough very fine ejection orifices.

In accordance with an embodiment of the invention, a liquid for ejectionincludes a bio-specimen and at least one kind of first compoundsexpressed by Formula (1):

wherein, in Formula (1), m≧8, and 8≦n≦18.

As a result, it becomes possible to prevent molecules of proteins andthe like contained in the bio-specimen from adhering to surfaces nearejection orifices and surfaces of flow paths of an ink jet head whichcauses clogging of the ejection orifices and the flow paths, therebysecuring stable ejection. Also, addition of these compounds in thebio-specimen does not lower the bioactivity of the biomoleculescontained in the bio-specimen, whereby a high level of reproducibilityof their biochemical reaction can be obtained.

The first compound may preferably be included by 1 weight % or more ofthe bio-specimen. As a result, sufficient ejection stability can beobtained even after a long time has elapsed since the liquid forejection is filled in the ink jet head.

Also, in Formula (1), it is preferred that m≧12, and n=12. As a result,sufficient ejection stability can be obtained even after a long time haselapsed since the liquid for ejection is filled in the ink jet head.

In accordance with an embodiment of the invention, a liquid for ejectionincludes a bio-specimen and at least one kind of seconds compoundsexpressed by Formula (2):

wherein, in Formula (2), m≧10, and 4≦n≦10.

As a result, it becomes possible to prevent molecules of proteins andthe like contained in the bio-specimen from adhering to surfaces nearejection orifices and surfaces of flow paths of an ink jet head whichcauses clogging of the ejection orifices and the flow paths, therebysecuring stable ejection. Also, addition of these compounds in thebio-specimen does not lower the bioactivity of the biomoleculescontained in the bio-specimen, whereby a high level of reproducibilityof their biochemical reaction can be obtained.

The second compound may preferably be included by 1 weight % or more ofthe bio-specimen. As a result, sufficient ejection stability of an inkjet head can be obtained.

Also, in Formula (2), it is preferred that m≧12, and n=6. As a result,sufficient ejection stability of an ink jet head can be obtained.

The bio-specimen may preferably include blood serum. By this, the amountof blood sampling required for examination can be substantially reduced.

According to a method for ejecting a bio-specimen in accordance with anembodiment of the invention, at least one kind of first compoundsexpressed by Formula (1) is added to a bio-specimen, and thebio-specimen is ejected by using an ink jet method.

wherein, in Formula (1), m≧8, and 8≦n≦18.

As a result, it becomes possible to prevent molecules of proteins andthe like contained in the bio-specimen from adhering to surfaces nearejection orifices and surfaces of flow paths of an ink jet head whichcauses clogging of the ejection orifices and the flow paths, therebysecuring stable ejection. Also, addition of these compounds in thebio-specimen does not lower the bioactivity of the biomoleculescontained in the bio-specimen, whereby a high level of reproducibilityof their biochemical reaction can be obtained.

According to a method for ejecting a bio-specimen in accordance withanother embodiment of the invention, at least one kind of secondcompounds expressed by Formula (2) is added to a bio-specimen, and thebio-specimen is ejected by using an ink jet method.

wherein, in Formula (2), m≧10, and 4≦n≦10.

As a result, it becomes possible to prevent molecules of proteins andthe like contained in the bio-specimen from adhering to surfaces nearejection orifices and surfaces of flow paths of an ink jet head whichcauses clogging of the ejection orifices and the flow paths, therebysecuring stable ejection. Also, addition of these compounds in thebio-specimen does not lower the bio-activity of the biomoleculescontained in the bio-specimen, whereby a high level of reproducibilityof their biochemical reaction can be obtained.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table showing evaluation results obtained for liquid forejection in accordance with an embodiment of the invention, in whichchanges in their ejection stability with elapsed time are evaluated.

FIG. 2 shows photographs capturing the moments when liquid for ejectionin accordance with the embodiment of the invention is ejected fromejection orifices.

FIG. 3 is a table showing results of measurement of the biochemicalreactivity of blood serum protein molecules contained in liquid forejection in accordance with the embodiment of the invention.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Preferred embodiments of the invention are described below. Liquid forejection in accordance with an embodiment of the invention may beobtained by adding a compound in a bio-specimen. The compound to beadded is a first compound expressed by Formula (1) below, or a secondcompound expressed by Formula (2) below. The first and second compoundsare straight-chain alkyl surfactant including ethylene glycol chain.

The bio-specimen may be blood, blood serum or the like. Here, humanblood serum sample CRPII (in which C-reactive protein is added in humanblood serum to a predetermined level of concentration) is used.

The first compounds are surfactant molecules including ethylene glycolchain (chain length m) and alkyl chain (chain length n). Here, m≧8, and8≦n≦18.

The second compounds are surfactant molecules including ethylene glycolchain (chain length m) and alkyl chain (chain length n). Here, m≧10, and4≦n≦10.

Table 1 below shows examples of the first and second compounds.

TABLE 1 Name of Compound Supply Source Molecular Structure of CompoundPEG12 Polypure n = 12, m = 12 in Formula (1) PEG24 Polypure n = 12, m =24 in Formula (1) PEG36 Polypure n = 12, m = 36 in Formula (1) P1777Tokyo Chemical n = 12, 8 ≦ 5 m ≦ 36 in Formula (1) Industry Co., Ltd.PEG-B-24 Polypure n = 6, m = 24 in Formula (2)

Examples of a method of synthesizing PEG36 among the first compounds aredescribed.

1) Synthesis of Dibenzyl-PEG36

As shown in Formula (3), dimethylate PEG 12 (7) and mono-benzyl PEG12(8) are reacted in the presence of bases (pH=12), thereby synthesizingdibenzyl-PEG36 (9).

2) Synthesis of Monobenzyl-PEG36

As shown in Formula (4), dibenzyl-PEG36 (9) is dissociated byhydrogenation reduction with palladium-carbon catalysis, and is furthersubject to heat reflux in a methanol solvent. Upon confirming thatdibenzyl-PEG36 is completely disappeared from the reaction mixedsolution by HPLC (High Performance Liquid Chromatography) or the like,the reaction product is purified and separated by column chromatography,thereby obtaining monobenzyl-PEG36 (10).

3) Synthesis of Benzyl-PEG36-Dodecane and PEG36-Dodecyl Ether (Formula(5))

Mono-benzyl-PEG36 (10) is dissolved in trifluorotoluene (TFT), anddodecyl bromide and potassium tert-butoxide (tert-BuOK) are dissolvedtherein, which is then subject to heat reflux. After completing thereaction, extraction and purification are conducted, thereby obtainingbenzyl-PEG36-dodecane (12). Thereafter, benzyl-PEG36-dodecane (12) isdissolved in methanol, and is dissociated by hydrogenation reductionwith palladium-carbon catalysis. After completing the reaction, thereaction product is extracted and purified by column chromatography,thereby obtaining white powder-like PEG36-dodecyl ether (13).

Next, a method of manufacturing the liquid for ejection is described.Here, the method is described using an example in which blood serum isused as the bio-specimen. First, blood sampled from a person to beexamined is set on a centrifugal separator, thereby separating bloodserum liquid that is the supernatant fluid.

Then, the first compound or the second compound described above is addedin 10 ml of the separated blood serum, and is dissolved in the bloodserum with as little shaking as possible. The addition amount of thecompound is desirably 1 weight % of the blood serum or higher. Thecompound is dissolved at room temperature. Alternatively, the compoundmay be dissolved, while lightly shaking a container containing themdipped in warmed water at 35° C. By this, the dissolving rate can beincreased. After dissolving, the separated and precipitated insolublesubstance is removed, and the solution is stored for half a day at 5° C.

Next, the liquid for ejection in accordance with the present embodimentwas filled in ink jet heads, and changes in their ejection stabilitywith elapsed time were evaluated, while changing the elapsed time fromcharging the liquid for ejection until discharging the same. FIG. 1shows the evaluation results.

FIG. 1 is a table showing the results of evaluation of the ejectionstability, obtained by changing the kind and amount (weight %) of thecompound added and the elapsed time from filling the liquid for ejectionuntil its ejection. The table in FIG. 1 shows how many of 12 nozzlesstably ejected the liquid. For example, when 10 nozzles among the 12nozzles performed stable ejection, this is expressed as (10/12).

Blood serum contains many molecules, such as, protein molecules thatwould likely adhere nonspecifically to surfaces near ejection orificesand flow paths of the ink jet head. Therefore, there are cases wherethose molecules, when adhered, clog the ejection orifices and the flowpaths. Also, as the time elapses, the blood serum liquid may dry andcoagulate, which may cause clogging of the ejection orifices and theflow paths.

Among the compounds added, P200, P400 and P11000 (which are all suppliedfrom Nippon Oil & Fats Co., Ltd.) are polyethylene glycol with the meanmolecule weight being 200, 400 and 11000, respectively. Thesepolyethylene glycol acts to prevent the blood serum liquid from drying.

As shown in FIG. 1, when P200, P400 and P11000 were added, the result of12/12 was obtained immediately after dissolving; but five seconds later,the result deteriorated to 3/12-0/12. This shows that the ejection wouldnot be stabilized by mere addition of polyethylene glycol.

Looking at the first compound (PEG12, PEG24, PEG36, P1777) and thesecond compound (PEG-B-24), it is observed that those of the compoundshaving longer chain length of hydrophilic ethylene glycol contained inthe molecules (compounds with greater m) maintain the ejection stabilityat high level even after a longer time has elapsed. As for the additionamount, the addition of 1 weight % or more is effective, but it isobserved that higher stability can be obtained when the addition amountis 3 weight % or higher.

FIG. 2 shows photographs capturing the moments when liquid for ejectionis ejected from ejection orifices. Three kinds of liquid, i.e., bloodserum with PEG36 added by 3 weight %, blood serum with PEG24 added by 3weight %, and blood serum alone, were prepared. FIG. 2 shows the statesof four or five samples in which the liquid for ejection was ejected 60seconds later after the liquid was filled in ink jet heads. As shown inthe figure, each of the photographs shows liquid ejected from 12nozzles. In the case of blood serum alone, it is observed that many ofthe trajectories of the ejected liquid are curved. This is becausemolecules such as protein molecules in the blood serum adherednonspecifically to surfaces near the ejection orifices and surfaces ofthe flow paths, whereby the ejection directions of the droplets werechanged. In this manner, when flight curving of droplets occurs, theamount of liquid to be ejected cannot be correctly controlled, such thatthe liquid for ejection cannot be correctly dispensed.

On the other hand, in the case of the blood serum with PEG36 added by 3weight % and the blood serum with PEG24 added by 3 weight %, nonspecificadhesion of molecules such as protein molecules can be prevented, suchthat flight curving of droplets does not occur, and therefore smooth andstable ejection becomes possible. Therefore, correct dispense of theliquid for ejection can be made possible.

Next, changes in the biochemical reactivity of biomolecules contained inthe liquid for ejection in accordance with the present embodiment wereevaluated, and FIG. 3 shows the evaluation results.

FIG. 3 is a table showing results of measurement of the biochemicalreactivity of various blood serum protein molecules. The table in FIG. 3shows differences (%) from a reference value that measures thebioactivity of blood serum alone, for the liquids in which PEG12, PEG24,PEG36, P1777, SF1, SF2, SF3 are dissolved in blood serum, respectively.Those having differences of ±5% or greater are shown in bold type. Inthe case of PEG36, the measurement was conducted with its concentrationchanged from 1 weight % to 4 weight %. In the cases of PEG12, PEG24 andP1777, their concentration was set to 3 weight %. For the liquids withPEG12, PEG24 and P1777, the results obtained by directly measuring thesolutions (dissolving), and the results obtained by measuring the liquidejected by the ink jet heads (head dispensing) are shown. It is notedthat the liquids with SF1, SF2 and SF3, and the liquid with PEG36 addedby 1 weight % and 2 weight % did not provide stable ejection, anddispense by the inkjet heads could not be performed.

SF1 and SF2 (supplied from Polypure) are compounds expressed by Formula(6) below. In Formula (6), n=12 in SF1, and n=24 in SF2.

SF3 (supplied from Tokyo Chemical Industry Co., Ltd.) is compoundexpressed by Formula (7) below. In Formula (7), n=12.

As shown in FIG. 3, in the cases of PEG12, PEG24, PEG36, P1777, it isobserved that those of the compounds having longer chain length ofhydrophilic ethylene glycol contained in the molecules (compounds withgreater m) are more effective in maintaining the bioactivity of proteinsin the blood serum. In particular, when PEG36 is added by 3 weight % ormore, high reproducibility of the biochemical reaction was observedexcept some of the proteins such as ALT and CRP.

As described above, according to the liquid for ejection in accordancewith the present embodiment, the first compound expressed by Formula (1)or the second compound expressed by Formula (2) is added in blood serumor the like by 1 weight % or more, whereby it becomes possible toprevent proteins contained in the blood serum from adhering to surfacesnear the ejection orifices and surfaces of the flow paths of ink jetheads which causes clogging of the ejection orifices and the flow paths,thereby securing stable ejection.

Also, addition of these compounds in blood serum does not lower thebioactivity of the biomolecules contained in the blood serum, whereby ahigh level of reproducibility of their biochemical reaction can beobtained, except some proteins.

Also, those of the first compounds and the second compounds havinglonger chain length of hydrophilic ethylene glycol contained in theirmolecules (with greater m) maintain the ejection stability at high leveleven after a longer time elapses. Also, the addition of 1 weight % ormore is effective, but higher stability can be obtained when theaddition amount is 3 weight % or higher.

What is claimed is:
 1. A liquid for ejection comprising: a bio-specimenthat includes blood serum; and at least one kind of first compoundsexpressed by Formula (1),

wherein: in Formula (1), m≧12 and n=12, and the liquid includes the atleast one kind of first compounds expressed by Formula (1) ranging from2 to 4 weight %.
 2. A liquid for ejection according to claim 1, whereinthe blood serum includes a plurality of proteins having bioactivity,wherein the bioactivity of the plurality of proteins is maintained inthe liquid for ejection.
 3. A liquid for ejection according to claim 2,wherein the plurality of proteins includes at least one protein selectedfrom the group consisting of C-Reactive Protein (CRP), Albumin (Alb),Lactate Dehydrogenase (LDH), Amylase (Amy), Creatinine (Cre), or acombination thereof.
 4. A liquid for ejection according to claim 1,wherein the liquid includes only one compound expressed by Formula (1).